Antisense Oligonucleotides
The proto-oncogene c-myb is the normal cellular homologue of the avian myeloblastosis virus-transforming gene v-myb. The c-myb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells. When the gene is altered in the appropriate manner, it has the potential to become an oncogene. Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
The human c-myb gene has been isolated, cloned, and sequenced. Majello et al, Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986).
C-myb is preferentially expressed in primitive hematopoietic tissues and hematopoietic tumor cell lines of several species. Westin et al., Proc. Natl. Acad. Sci. U.S.A. 79 2194 (1982). As cells mature, c-myb expression declines. Duprey et al., Proc. Natl. Acad. Sci. U.S.A. 82, 6937, (1985). The constitutive expression of exogenously introduced c-myb inhibits the erythroid differentiation of a murine erythroleukemia cell line (MEL) in response to known inducing agents. Clarke et al., Mol. Cell. Biol. 8, 884-892 (Feb. 1988). Although these data may implicate the c-myb gene product as a potentially important regulator of hematopoietic cell development, this evidence is largely of an indirect nature.
Some investigators report that c-myb may play an important role in regulating hematopoietic cell proliferation, and perhaps differentiation, Slamon et al, Science 233, 347 (1986); Westin et al, supra; Duprey et al, supra. The function of the c-myb proto-oncogene in normal hematopoiesis remains speculative.
Expression of specific genes may be suppressed by oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the target gene. This "antisense" methodology finds utility as a molecular tool for genetic analysis. Antisense oligonucleotides have been extensively used to inhibit gene expression in normal and abnormal cells in studies of the function of various proto-oncogenes.
Proliferation of the human promyelocytic leukemia cell line HL-60, which over-expresses the c-myc proto-oncogene, is inhibited in a sequence-specific, dose-dependent manner by an antisense oligodeoxynucleotide directed against a predicted hairpin loop containing the initiation codon of human c-myc. Wickstrom et al, Proc. Natl. Acad. Sci. USA 85, 1028-1032 (Feb. 1988). Inhibition of c-myc expression and/or cell proliferation in HL-60 or other cells by c-myc antisense oligonucleotides is described by the following: Loke et al, Clin. Res. 36 (3), 443A (abstract) (1988); Holt et al, Mol. Cell. Biol. 8, 963-973 (Feb. 1988); Yakoyama et al, Proc. Natl. Acad. Sci. U.S.A. 84, 7363-7367 (Nov. 1987); Harel-Bellan et al, J. Immunol. 140, 2431-2435 (Apr. 1988) and J. Cell. Biochem. Supplement 12A, 167 (Jan. 1988).
Antisense methodology has been used to study the expression of c fos, another proto-oncogene. C fos expression and cell transition from G.sub.0 to renewed growth is inhibited in 3T3 fibroblast cells transformed with an antisense oligodeoxynucleotide to the proto-oncogene. Nishikura et al, Mol. Cell. Biol. 7, 639-649 (Feb. 1987) and J. Cell. Biochem. Supplement 11A-D, 146 (1987). Also see Riabowol et al, Mol. Cell. Biol. 8, 1670-1676 (April 1988).
Mercola et al, Biochem. Biophys. Res. Comm. 147, 288-294 (Aug. 1987) disclose transfection of v-sis transformed cells with a plasmid directing expression of antisense c-fos RNA. The transfected cells exhibited a decrease in growth.
Groger et al., Proceedings American Assn. for Cancer Research 29, 439 (March 1988) report inhibition of c fos expression in both transformed and non-transformed human hematopoietic cells by an Epstein Barr virus episomal vector containing c-fos antisense RNA.
Transfection of transformed MethA fibroblast and non-transformed 3T3 cells by antisense RNA to the oncogene p53 has resulted in reduction of growth rate and cell proliferation. Shohat et al., Oncogene 1, 277-283 (1987).
Penno et al., American Journal of Human Genetics 39 (3), Supplement, A38 (1986) report inhibition of Y1 mouse adrenal carcinoma cell growth after transfection with a plasmid directing antisense to the Ki-ras oncogene.
Reed et al., J. Cell. Biochem. Supplement 12A, 172, (Jan. 1988) report inhibition of leukemic B cells and normal peripheral blood lymphocytes with antisense oligonucleotides to bcl-2, a gene suggested to have oncogenic potential.
U.S. Pat. No. 4,689,320 discloses inhibition of viruses using antisense oligodeoxynucleotides as anti-viral agents.
While the antisense methodology is a useful tool for genetic analysis, TIG, Jan. 1985, p.22-25, antisense oligonucleotides have not been used as anti-tumor agents in practical applications. Moreover, there have been no reports of antineoplastic agents utilizing antisense oligonucleotides complementary to c-myb mRNA.